Preimplantation genetic diagnosis (PGD) of monogenic autosomal hereditary disorders following assisted conception usually involves the removal of one or two blastomeres from preimplantation embryos. However, the amount of DNA from a single blastomere is insufficient to amplify the region of interest. Hence, the whole genome amplification (WGA) method is performed prior to amplifying the genes of interest before analysis of DNA material through polymerase chain reaction (PCR).
The present investigation was carried out in an attempt to study the phylogenetic analysis of different breeds of domestic chickens in Peninsular Malaysia inferred from partial cytochrome b gene information and random amplified polymorphic DNA (RAPD) markers. Phylogenetic analysis using both neighbor-joining (NJ) and maximum parsimony (MP) methods produced three clusters that encompassed Type-I village chickens, the red jungle fowl subspecies and the Japanese Chunky broilers. The phylogenetic analysis also revealed that majority of the Malaysian commercial chickens were randomly assembled with the Type-II village chickens. In RAPD assay, phylogenetic analysis using neighbor-joining produced six clusters that were completely distinguished based on the locality of chickens. High levels of genetic variations were observed among the village chickens, the commercial broilers, and between the commercial broilers and layer chickens. In this study, it was found that Type-I village chickens could be distinguished from the commercial chickens and Type-II village chickens at the position of the 27th nucleotide of the 351 bp cytochrome b gene. This study also revealed that RAPD markers were unable to differentiate the type of chickens, but it showed the effectiveness of RAPD in evaluating the genetic variation and the genetic relationships between chicken lines and populations.
The genetic make-up of five populations of Oreochromis spp. was examined by microsatellite analysis. Eleven polymorphic microsatellite loci showed significant departures from the Hardy-Weinberg equilibrium. The mean heterozygosity ranged from 0.6280 to 0.7040 for each population. The genetic distance values showed a clear separation between O. niloticus and O. mossambicus. The differentiation of the O. niloticus populations was then tested with various genetic measures, which are based on both the Infinite Allele and the Stepwise Mutation models. All these measures grouped the populations similarly.
Molecular analysis has been performed on a Malaysian patient with a severe bleeding disorder due to factor XIII(A) subunit deficiency. Total mRNA was isolated from the patient's leucocytes and four overlapping segments corresponding to the entire coding region of the A subunit cDNA were amplified by RT-PCR. The cDNA segments amplified efficiently and were of expected size. Direct sequencing of the complete reading frame revealed a single homozygous base change (nt 1327G-T) in exon 10 corresponding to a missense mutation, Val414Phe, in the catalytic core domain of the A subunit monomer. The mutation eliminates a BsaJ1 restriction site and family screening showed that both parents were heterozygous for the defect. The base substitution was absent in 55 normal individuals. Val414 is a highly conserved residue in the calcium-dependent transglutaminase enzyme family. Computer modelling based on 3D crystallographic data predicts that the bulky aromatic side chain of the substituted phenylalanine residue distorts protein folding and destabilizes the molecule. In addition, conformation changes in the adjacent catalytic and calcium binding regions of the A subunit are likely to impair the enzymatic activity of any protein synthesized.
Alpha-globin genes in crab-eating macaques were found to be triplicated at high frequencies according to restriction-enzyme comparisons. The frequencies of triplicated alpha-globin genes in macaques originally from Malaysia and Indonesia were 0.432 and 0.275, respectively, while no triplication was found in individuals from either the Philippines or northern and central Thailand. Quadruplicated alpha-globin genes were also observed, at frequencies of 0.045 (Malaysia), 0.075 (Indonesia), and 0.021 (the Philippines). A single locus was detected in only one of 40 chromosomes from Indonesia (frequency 0.025).
Telomeres shorten with physiological aging but undergo substantial restoration during cancer immortalization. Increasingly, cancer studies utilize the archive of formalin-fixed, paraffin-embedded (FFPE) tissues in diagnostic pathology departments. Conceptually, such studies would be confounded by physiological telomere attrition and loss of DNA integrity from prolonged tissue storage. Our study aimed to investigate these two confounding factors. 145 FFPE tissues of surgically-resected, non-diseased appendixes were retrieved from our pathology archive, from years 2008 to 2014. Cases from 2013 to 2014 were categorized by patient chronological age (0-20 years, 21-40 years, 41-60 years, > 60 years). Telomere lengths of age categories were depicted by telomere/chromosome 2 centromere intensity ratio (TCR) revealed by quantitative fluorescence in situ hybridization. Material from individuals aged 0-20 years from years 2013/2014, 2011/2012, 2009/2010, and 2008 were compared for storage effect. Telomere integrity was assessed by telomere fluorescence intensity (TFI). Epithelial TCRs (mean ± SD) for the respective age groups were 4.84 ± 2.08, 3.64 ± 1.21, 2.03 ± 0.37, and 1.93 ± 0.45, whereas corresponding stromal TCRs were 5.16 ± 2.55, 3.84 ± 1.36, 2.49 ± 1.20, and 2.93 ± 1.24. A trend of inverse correlation with age in both epithelial and stromal tissues is supported by r = -0.69, p < 0.001 and r = -0.42, p < 0.001 respectively. Epithelial TFIs (mean ± SD) of years 2013/2014, 2011/2012, 2009/2010 and 2008 were 852.60 ± 432.46, 353.04 ± 127.12, 209.24 ± 55.57 and 429.22 ± 188.75 respectively. Generally, TFIs reduced with storage duration (r = -0.42, p < 0.001). Our findings agree that age-related telomere attrition occurs in normal somatic tissues, and suggest that an age-based reference can be established for telomere studies on FFPE tissues. We also showed that FFPE tissues archived beyond 2 years are suboptimal for telomere analysis.
Gelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus' consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.
The inherent ability of nucleic acids to recognize a complementary pair has gained wide popularity in DNA sensor applications. DNA molecules can be produced in bulk and easily incorporated with various nanomaterials for sensing applications. More complex designs and sophisticated DNA sensors have been reported over the years to allow DNA detection in a faster, cheaper, and more convenient manner. Here, we report a DNA sensor designed to function like a switch to turn "on" silver nanocluster (AgNC) generation in the presence of a specific DNA target. By defining the probe region sequence, we are able to tune the color of the AgNC generated in direct relation to the different targets. As a proof of concept, we used dengue RNA-dependent RNA polymerase conserved sequences from all four serotypes as targets. This method was able to distinguish each dengue serotype by generating the serotype-respective AgNCs. The DNA switch was also able to identify and amplify the correct target in a mixture of targets with good specificity. This strategy has a detection limit of between 1.5 and 2.0 µM depending on the sequence of AgNC. The DNA switch approach provides an attractive alternative for single-target or multiplex DNA detection.
Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.
The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment.
A 26-year-old Chinese-Malaysian female patient with beta-thalassemia is presented. The main hematological values found in this patient were as follows: 1) normocytic hypochromic anemia (RBC 444 x 10(4)/microliters, Hb 11.8 g/dl) with marked anisopoikilocytosis, 2) erythroid hyperplasia, and 3) increased HbF (HbA 41.4%, HbA2 2.9%, HbF 48.9%). DNA obtained from peripheral leukocytes was analyzed using dot blot hybridization of the polymerase chain reaction (PCR)-amplified DNA with allele-specific oligonucleotide probes. A C----T substitution at position 654 of the second intervening sequence (IVS-2) was detected in her beta-globin clone.
We report a highly sensitive and selective multiplex assay by empowering an electrochemical DNA sensor with isothermal rolling circle amplification. The assay could simultaneously detect and discriminate three common entero-pathogens in a single reaction, with femtomolar sensitivity. It is useful for field- or resource-limited settings.
The reproductive success of male primates is not always associated with dominance status. For example, even though male orangutans exhibit intra-sexual dimorphism and clear dominance relationships exist among males, previous studies have reported that both morphs are able to sire offspring. The present study aimed to compare the reproductive success of two male morphs, and to determine whether unflanged males sired offspring in a free-ranging population of Bornean orangutans, using 12 microsatellite loci to determine the paternity of eight infants. A single flanged male sired most of the offspring from parous females, and an unflanged male sired a firstborn. This is consistent with our observation that the dominant flanged male showed little interest in nulliparous females, whereas the unflanged males frequently mated with them. This suggests that the dominant flanged male monopolizes the fertilization of parous females and that unflanged males take advantage of any mating opportunities that arise in the absence of the flanged male, even though the conception probability of nulliparous females is relatively low.
Species substitution, the use of a low value fish in place of a high value fish, is the biggest problem in international trade and the leading cause of fraud in the fisheries arena sector. Current DNA barcoding systems have partly solved this problem but also failed in many instances to amplify PCR targets from highly processed products because of the degradation of a longer barcode marker (~650bp). In the present study, a novel mini barcode marker (295bp) was developed to discriminate fish species in raw and processed states forms. The barcode primers were cross-tested against 33 fish species and 15 other animal species and found to be universal for all the tested fish varieties. When 20 commercial fish products of five different categories were screened, all commercial fish sample yielded positive bands for the novel fish barcode. PCR product was sequenced to retrieve the species IDs that reflected 55% (11/20) of Malaysian fish products were mislabeled.
The exciting discovery of the semiconducting-like properties of deoxyribonucleic acid (DNA) and its potential applications in molecular genetics and diagnostics in recent times has resulted in a paradigm shift in biophysics research. Recent studies in our laboratory provide a platform towards detecting charge transfer mechanism and understanding the electronic properties of DNA based on the sequence-specific electronic response, which can be applied as an alternative to identify or detect DNA. In this study, we demonstrate a novel method for identification of DNA from different shrimp viruses and bacteria using electronic properties of DNA obtained from both negative and positive bias regions in current-voltage (I-V) profiles. Characteristic electronic properties were calculated and used for quantification and further understanding in the identification process. Aquaculture in shrimp industry is a fast-growing food sector throughout the world. However, shrimp culture in many Asian countries faced a huge economic loss due to disease outbreaks. Scientists have been using specific established methods for detecting shrimp infection, but those methods do have their significant drawbacks due to many inherent factors. As such, we believe that this simple, rapid, sensitive and cost-effective tool can be used for detection and identification of DNA from different shrimp viruses and bacteria.
With the development of new metagenomic techniques, the microbial community structure of common bed bugs, Cimex lectularius, is well-studied, while information regarding the constituents of the bacterial communities associated with tropical bed bugs, Cimex hemipterus, is lacking. In this study, the bacteria communities in the blood-fed and starved tropical bed bugs were analysed and characterized by amplifying the v3-v4 hypervariable region of the 16S rRNA gene region, followed by MiSeq Illumina sequencing. Across all samples, Proteobacteria made up more than 99% of the microbial community. An alpha-proteobacterium Wolbachia and gamma-proteobacterium, including Dickeya chrysanthemi and Pseudomonas, were the dominant OTUs at the genus level. Although the dominant OTUs of bacterial communities of blood-fed and starved bed bugs were the same, bacterial genera present in lower numbers were varied. The bacteria load in starved bed bugs was also higher than blood-fed bed bugs.
BACKGROUND: Apolipoprotein E (apoE) is encoded by a polymorphic gene located on chromosome 19. The three common apoE alleles are epsilon2, epsilon3 and epsilon4. We studied the frequencies of the apoE alleles and genotypes in the three ethnic groups-Malay, Chinese and Indian-in Malaysia using DNA amplification followed by agarose gel electrophoresis.
METHODS: EDTA blood was collected and DNA was extracted using proteinase K-SDS digestion and purified by phenol-chloroform extraction. The apoE gene sequence was amplified using the PCR and apoE genotyping was performed by restriction enzyme digestion with HhaI.
RESULTS: Genotyping of the apoE gene produces six genotypes-E2/E2, E2/E3, E3/E3, E2/E4, E3/E4 and E4/E4. The most common apoE genotype in the Malays, Chinese and Indians studied was E3/E3, thus the most common apoE allele was epsilon3. The three common apoE genotypes were E3/E3 followed by E3/E4 and E2/E3, except in the Indians where E2/E3 was not detected. The three apoE alleles were confirmed in the Malays, Chinese and Indians except for the epsilon2 allele which was absent in the Indians.
CONCLUSION: The combined frequency of the apoE alleles in the Malays, Chinese and Indians was 0.058, 0.829 and 0.114 for epsilon2, epsilon3 and epsilon4, respectively.
Plasmodium knowlesi is the main cause of malaria in Sarawak, where studies on vectors of P. knowlesi have been conducted in only two districts. Anopheles balabacensis and An. donaldi were incriminated as vectors in Lawas and An. latens in Kapit. We studied a third location in Sarawak, Betong, where of 2169 mosquitoes collected over 36 days using human-landing catches, 169 (7.8%) were Anopheles spp. PCR and phylogenetic analyses identified P. knowlesi and/or P. cynomolgi, P. fieldi, P. inui, P. coatneyi and possibly novel Plasmodium spp. in salivary glands of An. latens and An. introlatus from the Leucosphyrus Group and in An. collessi and An. roperi from the Umbrosus Group. Phylogenetic analyses of cytochrome oxidase subunit I sequences indicated three P. knowlesi-positive An. introlatus had been misidentified morphologically as An. latens, while An. collessi and An. roperi could not be delineated using the region sequenced. Almost all vectors from the Leucosphyrus Group were biting after 1800 h but those belonging to the Umbrosus Group were also biting between 0700 and 1100 h. Our study incriminated new vectors of knowlesi malaria in Sarawak and underscores the importance of including entomological studies during the daytime to obtain a comprehensive understanding of the transmission dynamics of malaria.
The Merbok Estuary comprises one of the largest remaining mangrove forests in Peninsular Malaysia. Its value is significant as it provides important services to local and global communities. It also offers a unique opportunity to study the structure and functioning of mangrove ecosystems. However, its biodiversity is still partially inventoried, limiting its research value. A recent checklist based on morphological examination, reported 138 fish species residing, frequenting or subject to entering the Merbok Estuary. In this work, we reassessed the fish diversity of the Merbok Estuary by DNA barcoding 350 specimens assignable to 134 species initially identified based on morphology. Our results consistently revealed the presence of 139 Molecular Operational Taxonomic Units (MOTUs). 123 of them are congruent with morphology-based species delimitation (one species = one MOTU). In two cases, two morphological species share the same MOTU (two species = one MOTU), while we unveiled cryptic diversity (i.e. COI-based genetic variability > 2%) within seven other species (one species = two MOTUs), calling for further taxonomic investigations. This study provides a comprehensive core-list of fish taxa in Merbok Estuary, demonstrating the advantages of combining morphological and molecular evidence to describe diverse but still poorly studied tropical fish communities. It also delivers a large DNA reference collection for brackish fishes occurring in this region which will facilitate further biodiversity-oriented research studies and management activities.